五步蛇毒血小板聚集抑制因子cDNA的克隆及表达

采用一步法抽提五步蛇毒腺总ＲＮＡ,通过ＲＴ－ＰＣＲ的扩增出低分子量金属蛋白酶酶原的ｃＤＮＡ,克隆并测定了全序列。根据推导的氨基酸序列,发现其中一个ｃＤＮＡ除编码一个低分子量金属蛋白酶外,羧基端还包括一个血小板聚集抑制因子,这一结果证实了蛇毒金属蛋白酶和血小板聚集抑制因子起源于蛇毒金属蛋白酶酶原的前体。     

两个菜花烙铁头蛇毒金属蛋白酶去整合素基因的克隆与序列分析(英文)

从菜花烙铁头蛇的毒腺中 ,利用RT PCR进行体外扩增 ,克隆到 2个金属蛋白酶 去整合素基因 ,命名为TJM 1、TJM 2 .TJM 1cDNA全长为 15 2 8bp ,编码 4 81个氨基酸 ;TJM 2cDNA全长为 15 78bp ,编码 4 84个氨基酸 .TJM 1和TJM 2都属于Ⅱ型蛇毒金属蛋白酶 ,由信号肽、前肽、金属蛋白酶、间隔肽和去整合素 5部分组成 .Ⅱ型蛇毒金属蛋白酶氨基酸序列的比较及进化分析显示 ,它可进一步分为两类 ,一类包括大多数Ⅱ型蛇毒金属蛋白酶 (其中含有TJM 1) ,而TJM 2和agkistin则组成了另一类 .并且TJM 2和agkistin的第 4 0 7位和第 4 2 6位残基都是半胱氨酸 ,而在其它Ⅱ型金属蛋白酶的相应位置 ,4 0 7位是丝氨酸 ,4 2 6位则缺失 .TJM 2和agkistin均有可与整合素α2 Ⅰ区域特异性结合的片段Q NRKRHDNAQ(残基 2 76～ 2 84 ) ,这个片段在其它Ⅱ型金属蛋白酶中并没有发现 .因此推断 ,TJM 2和agkistin可能属于一类新型的Ⅱ型蛇毒金属蛋白酶 .     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒 ,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库 ,从中克隆了金属蛋白酶 解整合蛋白Ussurin ,并进行了序列分析。结果显示 ,Ussurin开框读码序列由 14 34bp组成 ,编码 4 78个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出 ,Ussurin最初的翻译产物是酶原前体 ;依次含有 18氨基酸组成的信号肽 ,171氨基酸组成的酶原区和由 2 89氨基酸组成的Ussurin(2 0 0氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和 73氨基酸组成的解整合蛋白结构域 )。Ussurin的金属蛋白酶结构域含有 3对二硫键 ;解整合蛋白结构域含有 6对二硫键和特征性RGD(精氨酸 甘氨酸 天冬氨酸 )结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶 解整合蛋白呈现高度同源性属于P Ⅱ。氨基酸序列blast比对发现 ,酶原区和解整链蛋白结构域呈现极高的同源性 ,而金属蛋白酶结构域却出现了极高的变异 ,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域     

中国长白山乌苏里蝮蛇金属蛋白酶解整合蛋白Ussurin基因克隆和序列分析

采用Clontech链转换建库试剂盒,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库,从中克隆了金属蛋白酶/解整合蛋白Ussurin,并进行了序列分析。结果显示,Ussurin开框读码序列由1434bp组成,编码478个氨基酸。由核苷酸顺序推导的氨基酸序列可以看出,Ussurin最初的翻译产物是酶原前体;依次含有18氨基酸组成的信号肽,171氨基酸组成的酶原区和由289氨基酸组成的Ussurin（200氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和73氨基酸组成的解整合蛋白结构域）。Ussurin的金属蛋白酶结构域含有3对二硫键;解整合蛋白结构域含有6对二硫键和特征性RGD(精氨酸甘氨酸天冬氨酸)结构。其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶/解整合蛋白呈现高度同源性属于P-Ⅱ。氨基酸序列blast比对发现,酶原区和解整链蛋白结构域呈现极高的同源性,而金属蛋白酶结构域却出现了极高的变异,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域。     

两个金环蛇蛇毒磷脂酶A2基因的克隆与序列报道

从广西产金环蛇（Bungarus fasciatus)毒腺中抽提总RNA,经mRNA纯化后构建了金环蛇毒腺cDNA文库,根据已发表的眼镜蛇科蛇毒磷脂酶A2基因序列中的保守区设计探针筛选克隆,得到2个磷脂酶A2基因,测定两者序列,其cDNA的阅读框均为435bp,编码145个氨基酸的磷脂酶A2前体,其中包括27个氨基酸组成的信号肽,118个氨基酸组成的成熟蛋白质,两者均属于第一类磷脂酶A2,其等电点经计算机软件推算分别为7．96和7．95。根据序列比较分析,这2个磷脂酶A2基因所编码的蛋白质序列结构均区别于已报道的金环蛇蛇毒磷脂酶A2,是2个新的金环蛇蛇毒磷酯酶A2,分别命名为金环蛇蛇毒磷脂酶A2Ⅰ（Bf-PLA2I)和金环蛇磷脂酶A2Ⅱ（Bf-PLA2Ⅱ)。     

双向电泳结合质谱初步分析蛇岛蝮蛇毒蛋白质组

采用荧光染料(Cy5)标记中国辽宁蛇岛蝮(Gloydius shedaoensis shedaoensis,GSS)蛇毒(snake venom,SV,GSS-SV)蛋白质,获得了该蛇毒的双向十二烷基硫酸钠聚丙烯酰胺凝胶电泳(2D SDS-PAGE)图谱,经DeCyder软件分析,分辨出1000多个蛋白点,分子量范围在10～150ku间,等电点在4～7的蛋白质点占78.8%。凝胶后染色(post-staining)采用蛋白荧光染料Deep Purple,选取的5个蛋白点经胶内酶解,产生的肽段经高效液相色谱-电喷雾串联质谱(high performance liquid chromatography/HPLC-electro-spray ionization tandem mass spectrometry/ESI-MS/MS,HPLC-ESI-MS/MS)进行序列测定,质谱数据经Sequest Bioworks软件分析,为蛇毒L-氨基酸氧化酶、金属蛋白酶、类凝血酶、纤溶酶原激活物和磷脂酶A2的同源蛋白。本研究采用的荧光标记2DSDS-PAGE结合HPLC-ESI-MS/MS的技术适于高通量研究蛇毒蛋白组成。     

一个东亚钳蝎蝎毒素BmTXKβ基因的克隆及其基因表达调控

从东亚钳蝎 (ButhusmartensiiKarsch ,BmK)毒腺组织cDNA文库中分离的长链钾通道毒素BmTXKβcDNA序列 ,克隆了BmTXKβ基因组序列 .BmTXKβ基因含有一个长度为 886bp的内含子 ,定位于BmTXKβ成熟肽中 ,与其它蝎毒素基因内含子定位于信号肽的基因结构不同 .并且 ,BmTXKβ基因的内含子特征也与其它蝎毒素基因不同 .研究结果从基因水平上证实了BmTXKβ是一个新的蝎毒素样肽 .以BmTXKβcDNA序列为探针与蝎基因组DNASouthern杂交出现 2条特异性杂交带 .杂交结果为蝎毒素基因可能通过DNA重排、多拷贝或多基因家族来调控基因表达提供了证据 .     

中华蜜蜂蜂毒镇静肽基因的cDNA克隆和表达

从中华蜜蜂 (Apisceranacerana)工蜂毒腺中快速抽提总RNA ,用RT PCR扩增得到大小约为2 5 0bp的cDNA片段 ,测序得到的片段长度为 2 34bp ,为蜂毒前镇静肽原 (preprosecapin)基因编码区的cDNA .以 3′RACE方法 ,扩增和测定了 3′端非编码区 2 19bp序列 .中蜂前镇静肽原cDNA序列与已报道的欧洲意蜂该基因cDNA序列具有 92 %同源性 ,氨基酸序列具有 87%同源性 .代表成熟肽镇静肽的最后 2 5个氨基酸序列 ,中蜂与意蜂同源性为 88% .3′端非编码区cDNA序列与欧洲意蜂序列有 73 1%同源性 .将中华蜜蜂蜂毒镇静肽成熟肽编码区与 3′非编码区部分克隆 ,构建了镇静肽与谷胱甘肽转移酶融合表达的载体pGEX AcSecapin .将载体转化大肠杆菌BL2 1(DE3)进行融合表达 .表达产物与抗GST抗体在 2 9kD处有很强的交叉反应 .大肠杆菌超声破碎后的上清液用SDS PAGE检测到表达的蛋白多为可溶性融合蛋白 ,通过亲和层析柱纯化和凝血酶的切割得到了镇静肽蛋白     

皖南尖吻蝮蛇毒中类去整合蛋白/富含半胱氨酸两结构域cDNA的克隆和表达

为了探讨出血毒金属蛋白酶结构功能关系 ,通过 RT- PCR方法 ,从皖南尖吻蝮蛇( Agkistrodon acutus)毒腺总 RNA中扩增得到编码 P- 型出血毒金属蛋白酶的完整类去整合蛋白和富含半胱氨酸两个结构域 c DNA( AA/DC) .它全长 964bp的 c DNA,开放阅读框架编码 2 1 6个氨基酸残基 ,序列比较分析表明它同来自 Bothrops jararaca的 jararhagin- C、来自 Crotalus atrox的 catrocollastatin- C有很高的同源性 .在类去整合蛋白结构域中 ,Ser- Glu- Cys- Asp( SECD)代替了去整合蛋白中相应部位的 Arg- Gly- Asp( RGD)三肽序列 .将编码区基因克隆入 p GEX- 2 T载体中 ,转化大肠杆菌 TG- 1 ,用 IPTG诱导表达 ,表达产物具有抑制胶原诱导的血小板凝集活性 ,但不抑制ADP诱导的血小板凝集 .该研究为进一步阐述蛇毒金属蛋白酶结构功能关系和药物开发奠定了基础 .     

蛇毒中的金属蛋白水解酶

从很早以前 ,人们就进行了探索蛇毒的奥秘。由于近代生物化学和分子生物学的快速进步 ,这为揭示蛇毒的本质及其作用机制 ,提供了有力条件。迄今为止 ,蛇毒被誉为自然界最集中的酶原之一 ,其中有功能独特的酶、毒性蛋白质和活性短肽等 ,目前已经在蛇毒中分离出至少 1 50种蛋白水解酶 ,这些蛋白酶可分为丝氨酸蛋白酶和金属蛋白酶 ,这里我们只讨论后者。蛇毒金属蛋白酶具有使纤维蛋白溶解的作用 ,其中有的酶具有底物专一性 ,只作用于凝血因子 X、血小板膜受体或 von Willebrand因子。很多蛇毒金属蛋白酶由多个结构域组成 ,如蛋白水解酶活性结…     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

蕲蛇蛇毒中一种新型金属蛋白酶AA-MP-I的纯化和表征

本研究通过嗜硫色谱、Sephadex G-75、蓝胶和POROS HQ20离子交换色谱,从蕲蛇蛇毒中分离得到一种新组分AA-MP-I。该酶为分子量22.9kDa的单体蛋白,等电点为5.55,不含中性糖基,N端序列为STE-FQRYMEIVIVVDHSMVK,结果表明其为新型P-I型金属蛋白酶,对温度敏感,具有抗凝血活性,40℃下抗凝血活性最强,具有出血毒性,无磷脂酶A2活性。     

Cloning, sequence analysis and expression in E. coli of the cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas

The cDNA of the thrombin-like enzyme (pallabin) from the venom of Agkistrodon halys pallas was cloned and sequenced. The length of the cDNA is 923bp which includes 120bp of noncoding region and 780bp of coding region. Pallabin was synthesized as a prozymogen with 260 amino acids, which includes a signal peptide of 18 amino acids, a proposed propeptide of 6 amino acids and a matured peptide of 236 amino acids. Pallabin exhibits a strong amino acid similarity to the serine proteases isolated from other snake venoms. It contains 12 cysteins which form 6 disulfide bridges. Like other serine proteases, it also has three conserved catalytically active sites: His41, Asp86 and Ser182. To our knowledge, this study is the first report concerning the cDNA of a thrombin-like enzyme from Agkistrodon halys pallas. The cDNA was cloned into the expression plasmid pT7ZZa and expressed in E.coli. The recombinant pallabin immunologically reacted with its specific antibody.     

Complete amino acid sequence of kaouthiagin, a novel cobra venom metalloproteinase with two disintegrin-like sequences

The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC(50) of 0.2 microM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S-S linkage. Both the synthetic cyclized peptides from the disintegrin-like domain and from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC(50) values of approximately 90 and approximately 4.5 microM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.     

Characterization and cDNA cloning of a platelet aggregation inhibitor

A novel platelet aggregation inhibitor, sal-C, was purified to homogeneity from the venom of Korean snake (Agkistrodon halys brevicaudus). Several lines of experimental evidence clearly indicated that sal-C inhibits not only the collagen-induced platelet aggregation, but also the aggregation mediated by the cell surface glycoprotein IIb-IIIa (GP IIb-IIIa). We have isolated the cDNA encoding sal-C from the cDNA library of the snake venom gland and analyzed its complete nucleotide sequence. Sal-C is a single-chain polypeptide composed of 212 amino acids including 24 cysteines. The deduced polypeptide sequence of sal-C demonstrated considerable homology to previously described protein species of the collagen-induced platelet aggregation inhibitor family. Sal-C does not have the Arg-Gly-Asp (RGD) motif, but contains the Ser-Glu-Cys-Asp sequence. Interestingly, sal-C was found to inhibit GP IIb-IIIa binding to immobilized fibrinogen which is antagonized by the typical RGD motif of disintegrins.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.